Host factor for RNA phage QB replicase (Hfq) is a global regulator that regulates sRNA-mRNA binding and stability and is involved in bacterial growth, chemotaxis, virulence, drug resistance, and response to external stress. The hfq gene of 59 clinical isolates of Klebsiella pneumoniae (KP) collected from Aug 2017 to Aug 2018 in our hospital were extracted and compared with the ones downloaded from NCBI database. Transeq, multiple sequence alignments of amino acid sequences were performed with MAFFT software, and the secondary structure of the protein were analyzed using the online website ESPript3.0. The physicochemical properties of the amino acid sequences were calculated using the online website ProtParam. Hfq domains were predicted from Conserved domains in the NCBI database. The tertiary structure of Hfq protein was predicted using Phyre2. The CRISPR/Cas9 homologous recombination technology was used to knock out the hfq gene in KP strain. The wild strain and the hfq knockout strain were diluted by 1∶100, and 0.1 μg/mL, 0.2 μg/mL, and 0.3 μg/mL Imipenem/cilastatin sodium were added to co-culture respectively. The growth curve was drawn by taking the 1 h, 2 h, 3 h, 4 h bacteria liquid count respectively. Despite 5 hfq gene mutations detected in 3 strains, the amino acid sequences of all strains were identical. The tertiary structure prediction indicated that 68 (66.67%) amino acids were consistent with the template sequence, and the more conserved functional domain was located at the 54-59 sites (VYKHAI). The hfq gene of one clinical strain was successfully knocked out by CRISPR/Cas9 homologous recombination. The resistance of the hfq knockout strain to antibiotic significantly decrease than that of the wild type(P<0.05). The results of this study suggest that KP’s Hfq amino acid sequence is conserved, and the gene may play a role in carbapenems resistance.